ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 292, No. 2, February 1, pp. 419-426, 1992 Cytochrome c2 Mutants of Rhodobacter capsulatus Michael Caffrey,*,’ Edgar Davidson,? Michael Cusanovich,* and Fevzi Daldal$ *Department of Biochemistcg, University of Arizona, Tucson, Arizona 85721; tlnstitute for Structural and Functional Studies, 3401 Mark Street, Philadelphia, Pennsylvania 19104; and $Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received June 17, 1991, and in revised form September 24,1991 Although structurally related to other members of the class I c-type cytochromes, the cytochromes cg have little amino acid sequence homology to the eukaryotic cyto- chromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cyto- chromes, we have develalped a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. cap- sulatus wild-type cytochrome c2 in cytochrome cz-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement 4or photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mu- tants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overprodu.ced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These flnd- ings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect de- fective cytochrome c2 mutants. 0 1992 Academic Press, Inc. With their well-established chemical and structural properties, the cytochromes c are among the best char- acterized proteins (1). In general, they are small, water- soluble molecules which a.re structurally, and often func- tionally, related to each other. A subclass of the class I c-type cytochromes, the cytochromes c2 from photosyn- thetic bacteria, functions as soluble electron carriers be- tween membrane-bound redox centers. They transfer 1 To whom correspondence should be addressed at present address: Centre d’Etudes Nucleaires, Laboratoire de Resonance Magnetique, 85X- 38041 Grenoble Cedex, France. 0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. electrons from the cytochrome bcr complex to the pho- tosynthetic reaction center under photosynthetic condi- tions (2) and to cytochrome oxidase under aerobic con- ditions (3). Cytochromes c2 have been purified from a large number of phototrophic bacteria, and the high-resolution structures of those from Rhodospirillum rubrum (4,5) and Rhodobacter capsulatus (6) have been determined. Al- though having substantial structural homology with the other class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology (less than 40%) and exhibit distinct properties. For example, the redox poten- tials of the cytochromes c2 range from 250 to 470 mV and their isoelectric points range from 4.5 to greater than 9. This is in sharp contrast to the eukaryotic cytochromes c which have redox potentials of 260 * 5 mV and are strongly basic (PI = 10.5). The development of a genetic system for the study of R. capsulatus cytochrome c2 by site-directed mutagenesis is of particular interest for a number of reasons. With respect to the eukaryotic cytochromes c, R. capsulatus cytochrome c2 has a relatively high redox potential of 367 mV at pH 7.0 and it is therefore interesting to examine the basis for this difference in redox potential (7). In ad- dition, the isoelectric point of R. capsulatus cytochrome c2 is near neutrality (PI = 7.1) which makes it amenable to studies characterizing electrostatic and steric inter- actions with other electron donors or acceptors (1). In contrast, the protein-protein interactions of the eukary- otic cytochromes c are dominated by a strong positive electrostatic field which complicates the interpretation of the electrostatic and steric effects of individual residues. Finally, the availability of the R. capsulatus cytochrome c2 three-dimensional structure provides the foundation for choosing residues to be mutated and aids in the in- terpretation of the resulting mutant properties (6). Importantly, the gene encoding R. capsulatus cyto- chrome c2 (cycA) has been cloned, and a cytochrome c2- minus strain of R. capsulatus prepared (8) which can serve as a host for the overexpression of mutants. It is well established that elimination of cytochrome c2 does not 419